Protein phosphorylation-mediated regulation of signaling, growth and transcription is not only key to normal cellular regulation, but also is commonly dysregulated in cancers. Many anti-cancer drugs are specifically directed at kinases. Protein GlcNAcylation (O-GlcNAc) is nearly as abundant as Ser(Thr) phosphorylation. O- GlcNAc cycles like phosphorylation, and is often competitive with it. O-GcNAc regulates signaling, transcription, and cytoskeletal functions in response to nutrients and stress. Recent glycoproteomic studies have revealed surprisingly extensive crosstalk or interplay between GlcNAcylation and phosphorylation. This crosstalk results not only from competition for occupancy at the same or proximal sites, but also by each modification regulating the activities of the other's enzymes. For example, O-GlcNAc Transferase is regulated by phosphorylation and kinases are regulated by GlcNAcylation. The goal of this study is to elucidate the global crosstalk between GlcNAcylation and phosphorylation at the individual site level and to understand the extent and mechanisms as to how GlcNAcylation regulates kinases. Aim 1: Elucidate the Dynamic Crosstalk Between GlcNAcylation and Phosphorylation. A. Using short-term treatments of HeLa cells with a general kinase inhibitor or a general phosphatase inhibitor, combined with novel quantitative glyco-proteomic methods, we will determine the changes in O-GlcNAc site occupancy on hundreds of proteins induced by a global increase or decrease in phosphorylation. These studies will not only reveal details of interplay, but will also identify many rapidly cycling O-GlcNAc sites on key regulatory proteins. B. A similar glyco-proteomic quantification of site occupancy will be performed to examine the affects of inhibiting single kinases (aurora kinase, mTOR, GSK32, Akt, PKC1) using specific inhibitors. Aim 2: Regulation of Kinases by GlcNAcylation. We will continue to identify GlcNAcylated kinases and study their regulation by O-GlcNAc, focusing initially on CAMKIV, PKC1, AKT, and Src. Aim 3: Study the Roles of O-GlcNAc in Cytokinesis. OGT transiently co-localizes with kinases, phosphatases, and O-GlcNAcase at the cell's mid-body during cytokinesis. Over-expression of OGT prevents cytokinesis causing polyploidy. We are elucidating the roles of the interplay of these enzymes and their substrates in regulating cytokinesis. Without a better understanding of the interplay between GlcNAcylation and phosphorylation, we will not understand the full picture of the regulation of signaling cascades that are key to cellular functions and that are dysregulated in cancer. Until recently, we have not had the technology required to study this interplay on a global level at individual sites. Data from these studies will set the foundation and lead to tools for future biological investigations that will lead to totally new avenues of therapeutic development.